1. Field of the Invention
The invention relates to a hepatitis C virus (HCV) cell culture system, which comprises mainly eukaryotic cells containing transfected HCV specific genetic material, which means they are transfected with HCV specific genetic material.
2. Background Information
The hepatitis C virus (HCV) is one of the main causes worldwide of chronic and sporadic liver diseases. The history of most HCV infections does not involve any obvious clinical signs, but 80-90% of the infected people become chronic carriers of the virus and 50% of these chronic carriers of the virus develop chronic hepatitis with different degrees of severity. Approx. 20% of the chronically infected develop a cirrhosis of the liver over 10 to 20 years, based on what a primary hepatocellular carcinoma can develop. Nowadays chronic hepatitis C is the main indication for liver transplantation. One currently available therapy involves high-dose administration of Interferon alpha or a combination of Interferon alpha and the purine nucleoside analogue Ribavirin. However, only approx. 60% of all treated persons respond to this therapy and with these, a new viraemia occurs in more than half of all cases after the discontinuation of the treatment.
Due to the high prevalence, especially in industrialized countries, the serious effects of chronic infections and the lack of effective therapy, the development of a HCV specific chemotherapy is an important goal of pharmaceutical research and development. Such a goal, however, has been hampered by the lack of a suitable cell culture system, which enables the study of virus replication and pathogenesis in eukaryotic cells.
Due to the small amount of virus in blood or tissue, the lack of suitable cell culture systems or animal models (the chimpanzee is still the only possible experimental animal) as well as the lack of efficient systems for producing virus-like particles, it has been difficult to analyze the molecular composition of the HCV particle in-depth. The information currently available can be summarized as follows: HCV is an enveloped plus-strand RNA virus with a particle diameter of 50-60 nm and a medium density of 1.03-1.1 g/ml. It was molecularly cloned and characterized for the first time in 1989 (Choo et al., 1989, Science, 244, 359-362). The HCV-RNA has a length of approx. 9.6 kb (=9600 nucleotides), a positive polarity and comprises one open reading frame (ORF), which encodes a linear polyprotein of approx. 3010 amino acids (see Rice 1996, in Virology, B. N. Fields, D. M. Knipe, P. M. Howley, Eds. (Lippincott-Raven, Philadelphia, Pa., 1996), vol. 1, pp. 931-960; Clarke 1997, J. Gen. Virol. 78, 2397; and Bartenschlager 1997, Intervirology 40, 378 and see FIG. 1A). During the replication of the virus the polyprotein is cleaved into the mature and functionally active proteins by cellular and viral proteases.
Within the polyprotein the proteins are arranged as follows (from the amino- to the carboxy terminus) : Core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The core protein is the main component of the nucleocapsid. The glycoproteins E1 and E2 are transmembrane proteins and the main components of the viral envelope. They probably play an important role during the attachment of the virus to the host cell. These three proteins core, E1, and E2 constitute the viral particle and are therefore called structural proteins. The function of the protein p7 is still not clear. The protein NS2 is probably the catalytic domain of the NS2-3 protease, which is responsible for the processing between the proteins NS2 and NS3. The protein NS3 has two functions, one is a protease activity in the amino terminal domain, which is essential for the polyprotein processing, and the other a NTPase/helicase function in the carboxy terminal domain, which is probably important during the replication of the viral RNA. The protein NS4A is a co-factor of the NS3 protease. The function of the protein NS4B is unknown.
The open reading frame is flanked on its 5xe2x80x2 end by a non-translated region (NTR) approx. 340 nucleotides in length, which functions as the internal ribosome entry site (IRES), and on its 3xe2x80x2 end by a NTR approx. 230 nucleotides in length, which is most likely important for the genome replication. A 3xe2x80x2to NTR such as this is the object of patent application PCT/US 96/14033. The structural proteins in the amino terminal quarter of the polyprotein are cleaved by host cell signal peptidase. The non-structural proteins (NS) 2 to (NS) 5B are processed by two viral enzymes, namely the NS2-3 and the NS3/4A protease. The NS3/4A protease is required for all cleavages beyond the carboxy terminus of NS3. The function of NS4B is unknown. NS5A, a highly phosphorylated protein, seems to be responsible for the Interferon resistance of various HCV genotypes (see Enomoto et al. 1995, J. Clin. Invest. 96, 224; Enomoto et al. 1996, N. Engl. J. Med. 334, 77; Gale Jr. et al. 1997, Virology 230, 217; Kaneko et al. 1994, Biochem. Biophys. Res. Commun. 205, 320; Reed et al., 1997, J. Virol. 71, 7187), and NS5B has been identified as the RNA-dependent RNA polymerase.
First diagnostic systems have been developed from these findings, which are either based on the detection of HCV specific antibodies in patient serum or the detection of HCV specific RNA using the reverse transcription polymerase chain reaction (RT-PCR), and which are routinely used with all blood and blood products and/or according to the regulations.
Since the first description of the genome in 1989 several partial and complete sequences of the HCV have been cloned and characterized using the PCR method. A comparison of these sequences shows a high variability of the viral genome in particular in the area of the NS5B gene, which eventually resulted in the classification of 6 genotypes, which are again subdivided into the subtypes a, b, and c.
The genomic variance is not evenly distributed over the genome. The 5xe2x80x2to NTR and parts of the 3xe2x80x2to NTR are highly conserved, while certain encoded sequences vary a lot, in particular the envelope proteins E1 and E2.
The cloned and characterized partial and complete sequences of the HCV genome have also been analyzed with regard to appropriate targets for a prospective antiviral therapy. In the course of this, three viral enzymes have been discovered, which may provide a possible target. These include (1) the NS3/4A protease complex, (2) the NS3 Helicase and (3) the NS5B RNA-dependent RNA polymerase. The NS3/4A protease complex and the NS3 Helicase have already been crystallized and their three-dimensional structure determined (Kim et al., 1996, Cell, 87,343; Yem et al., 1998, Protein Science, 7, 837; Love et al., 1996, Cell, 87, 311; Kim et al., 1998, Structure, 6, 89; Yao et al., 1997, Nature Structural Biology, 4, 463, Cho et al., 1998, J. Biol. Chem., 273, 15045). it has not been successful until now with the NS5B RNA-dependent RNA polymerase.
Even though important targets for the development of a therapy for chronic HCV infection have been defined with these enzymes and even though a worldwide intensive search for suitable inhibitors is ongoing with the aid of rational drug design as well as high throughput screening, the development of a therapy has one major deficiency, namely the lack of cell culture systems or simple animal models, which allow direct, reliable identification of HCV-RNA or HCV antigens with simple methods which are common in the laboratory. The lack of these cell culture systems is also the main reason that to date the comprehension of HCV replication is still incomplete and mainly hypothetical.
Although it has been reported that a close evolutionary relationship exists between HCV and the flavi- and pestiviruses, and self-replicating RNAs have been described for these, which can be used for the replication in different cell lines with a relatively high yield, (see Khromykh et al., 1997, J. Virol. 71, 1497; Behrens et al., 1998, J. Virol. 72, 2364; Moser et al., 1998, J. Virol. 72, 5318), similar experiments with HCV have not been successful to date.
Although it is known from different publications that cell lines or primary cell cultures can be infected with high titre patient serum containing HCV, (Lanford et al. 1994, Virology 202, 606; Shimizu et al. 1993, Proceedings of the National Academy of Sciences, USA, 90, 6037-6041; Mizutani et al. 1996, Journal of Virology, 70, 7219-7223; M. Ikeda et al. 1998, Virus Res. 56, 157; Fournier et al. 1998, J. Gen. Virol. 79, 2376 and bibliographical references quoted in here; Ito et al. 1996, Journal of General Virology, 77, 1043-1054), these virus-infected cell lines or cell cultures do not allow the direct detection of HCV-RNA or HCV antigens. The viral RNA in these cells can not be detected in a Northern Blot (a standard method for the quantitative detection of RNA) or the viral protein in a Western Blot or with immunoprecipitation. It has only been possible to detect HCV replication with very costly and indirect methods. These disadvantageous facts show that the replication in these known virus-infected cell lines or cell cultures is insufficient. Furthermore it is known from the publications of Yoo et al. (1995, Journal of Virology, 69, 32-38) and of Dash et al., (1997, American Journal of Pathology, 151, 363-373) that hepatoma cell lines can be transfected with synthetic HCV-RNA, which are obtained through in vitro transcription of the cloned HCV genome. In both publications the authors started from the basic idea that the viral HCV genome is a plus-strand RNA functioning directly as mRNA after being transfected into the cell, permitting the synthesis of viral proteins in the course of the translation process, and so new HCV particles are (could be) formed. This viral replication, which means these newly formed HCV viruses and their RNA, have been detected through RT-PCR. However the published results of the RT-PCR carried out indicate, that the HCV replication in the described HCV transfected hepatoma cells is not particularly efficient and is not sufficient to measure the quality, let alone the quantity of the fluctuations in the replication rate after an targeted action with prospective antiviral treatments. Furthermore, Yanagi et al., Proc. Natl. Acad. Sci. USA, 96, 2291-95, 1999 reports that the highly conserved 3xe2x80x2to NTR is essential for the virus replication. This knowledge contradicts the statements of Yoo et al. and Dash et al., who used for their experiments only HCV genomes with shorter 3xe2x80x2to NTRs since they did not know the authentic 3xe2x80x2 end of the HCV genome.
An object of the present invention is to provide a HCV cell culture system, where the viral RNA self-replicates in the transfected cells with such a high efficiency that the quality and quantity of the fluctuations in the replication rate can be measured with common methodologies usually found in the laboratory after a targeted action with virus and prospective HCV specific antivirals in particular.
The present invention is directed to a hepatitis C virus (HCV) cell culture system, which comprises mainly eukaryotic cells containing transfected HCV specific genetic material, characterized in that, the eukaryotic cells are human hepatoma cells and the transfected HCV specific genetic material is a HCV-RNA construct, which comprises the HCV specific RNA segments 5xe2x80x2to NTR, NS3, NS4A, NS4B, NS5A, NS5B, and 3xe2x80x2to NTR as well as an additional marker gene for selection (selection gene).
Thus, the present invention provides a cell culture system, where the eukaryotic cells are human cells, in particular hepatoma cells, which are preferably derived from a normal hepatoma cell line, but can also be obtained from an appropriate primary cell culture, and where the transfected HCV specific genetic material is a HCV-RNA construct, which essentially comprises the HCV specific RNA segments 5xe2x80x2to NTR, NS3, NS4A, NS4B, NS5A, NS5B, and 3xe2x80x2to NTR preferably in the order mentioned as well as a minimum of one marker gene for selection (selection gene).
Here and in the following xe2x80x9cNTRxe2x80x9d stands for xe2x80x9cnon-translated regionxe2x80x9d and is a known and familiar term or abbreviation in the relevant art.
Here and in the following the term xe2x80x9cHCV-RNA constructxe2x80x9d comprises constructs, which include the complete HCV genome, as well as those, which only include a part of it, which means a HCV subgenome.
A preferred embodiment of the cell culture system according to the invention, which had proven to be worthwhile in practice, is lodged at the DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of Microorganisms and Cell Cultures) in Braunschweig, Germany under the number DSM ACC2394 (laboratory name HuBl 9-13), with a date of deposit on Mar. 24, 1999.
With the cell culture system according to the invention an in vitro system is provided, where HCV-RNA is self-replicated and expressed intracellularly and in a sufficient amount, so that the quantity of the amounts of HCV-RNA as well as the HCV specific proteins can be determined with conventional and reliably precise biochemical measuring methods. The present invention thus provides a cell-based HCV replication system that is useful for, e.g., the development and testing of antiviral drugs. This test system may be used to identify potential targets for an effective HCV specific therapy and developing and evaluating HCV specific chemotherapeuticals.
The invention is based on the surprising finding that efficient replication of the HCV-RNA only occurs in cells if they have been transfected with an HCV-RNA construct, which comprises at least the 5xe2x80x2 and the 3xe2x80x2 non-translated regions (NTR) and the non-structural proteins (NS) 3 to 5B and additionally a marker gene for selection (selection gene). The structural genes are without great importance for replication, whereas efficient replication of the HCV-RNA apparently only occurs if the transfected cells are subject to permanent selection pressure, which is imparted by the marker gene for selection (selection gene) linked to the HCV-RNA. Consequently the marker gene (selection gene) seems on one hand to provoke the selection of those cells, where the HCV-RNA replicates productively, and it seems on the other hand to considerably increase the efficiency of the RNA replication.
An object of the invention is also a cell-free HCV-RNA construct, characterized in that it comprises the HCV specific RNA segments 5xe2x80x2to NTR, NS3, NS4A, NS4B, NS5A, NS5B, and 3xe2x80x2to NTR, preferably in the order mentioned, as well as a marker gene for selection (selection gene).
In the present context the terms 5xe2x80x2to NTR and NS3 and NS4A and NS4B and NS5A and NS5B and 3xe2x80x2to NTR comprise each nucleotide sequence, which is described in the state of the art as the nucleotide sequence for each functional segment of the HCV genome.
The HCV-RNA construct of the present invention may be used in conducting a detailed analysis of the HCV replication, pathogenesis and evolution in cell culture. The HCV specific viral RNA can specifically be created as a complete genome or subgenome in any amount, and it is possible, to manipulate the RNA construct and consequently to examine and identify the HCV functions on a genetic level.
Because all HCV enzymes identified as a main target for a therapy at the moment, namely the NS3/4A protease, the NS3 helicase and the NS5B polymerase, are included in the HCV-RNA construct according to the invention, it can be used for all relevant analyses.
An embodiment of the HCV-RNA construct, which has proven to be worthwhile in practical use, stands out by the fact that it comprises the nucleotide sequence according to the sequence protocol SEQ ID NO:1.
Further embodiments with similar good properties for practical use are characterized in that they comprise a nucleotide sequence either according to sequence protocol SEQ ID NO: 4-6 or according to sequence protocol SEQ ID NO: 7-9 or according to sequence protocol SEQ ID NO: 10-12 or according to sequence protocol SEQ ID NO: 13-15 or according to sequence protocol SEQ ID NO: 16-18 or according to sequence protocol SEQ ID NO: 19-21 or according to sequence protocol SEQ ID NO: 22-24 or according to sequence protocol SEQ ID NO: 25-27 or according to sequence protocol SEQ ID NO: 28-30 or according to sequence protocol SEQ ID NO: 31-33.
In certain embodiments, the HCV subgenomic construct comprises a 3xe2x80x2to NTR, which has a nucleotide sequence selected from the group of nucleotide sequences (a) to (i) listed in the following:
(a) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTT TTTTTAGCTTTTTTTTTTTTCTTTTTTTTTGAGAGAGAGAGTCTCACTCTGTTGC CCAGACTGGAGT (SEQ ID NO: 43)
(b) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTTTTTTTAGTC TTTTTTTTTTC TTTTTTTTGA GAGAGAGAGT CTCACTCTGT TGCCCAGACT GGAGC (SEQ ID NO: 44)
(c) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTT TTTAATCTTT TTTTTTTTCT TTTTTTTTGA GAGAGAGAGT CTCACTCTGT TGCCCAGACT GCAGC (SEQ ID NO: 45)
(d) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTTTTTTTTAGTC TTTTTTTTTT TCTTTTTTTT TGAGAGAGAG AGTCTCACTC TGTTGCCCAG ACTGGAGT (SEQ ID NO: 46)
(e) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTT TTTTTAGTCT TTTTTTTTTT TCTTTTTTTT TGAGAGAGAG AGTCTCACTC TGTTGCCCAG ACTGGAGT (SEQ ID NO: 47)
(f) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTTTTTTTAGTCT TTTTTTTTTT TCTTTTTTTT TTGAGAGAGA GAGTCTCACT CTGTTGCCCA GACTGGAGT (SEQ ID NO: 48)
(g) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTTTTTTTAGTCT TTTTTTTTTT CTTTTTTTTT GAGAGAGAGA GTCTCACTCT GTTGCCCAGA CTGGAGT (SEQ ID NO: 49)
(h) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTTTTTTTTTAAT CTTTTTTTTT TTTTTCCTTT TTTTGAGAGA GAGAGTCTCA CTCTGTTGCC CAGACTGGAG T (SEQ ID NO: 50).
(i) ACGGGGAGCTAAACACTCCAGGCCAATAGGCCATCCTGTTTTTTTTTTAATC TTTTTTTTTT TTTTCTTTTT TTTTTGAGAG AGAGAGTCTC ACTCTGTTGC CCAGACTGGAGT(SEQ ID NO: 51)
The marker gene for selection (selection gene) included in the HCV-RNA constructs according to the invention is preferably a resistance gene, in particular an antibiotic resistance gene.
This has the advantage that the cells transfected with this construct can easily be selected from the non-transfected cells by adding for example the appropriate antibiotic to the cell culture medium in the case of an antibiotic resistance gene.
In the present context xe2x80x98antibioticxe2x80x99 means any substance, which impedes the non-transfected host cells or the cells, where the HCV-RNA is not replicating efficiently, continuing to live or grow, especially the cell poison Puromycin, Hygromycin, Zeocin, Bleomycin or Blasticidin.
A preferred marker gene for selection (selection gene) and resistance gene, which has proven to be worthwhile in practice, is the neomycin phosphotransferase gene.
An alternative for the antibiotic resistance genes is for example the thymidine kinase gene, which can be used to carry out a HAT selection.
The marker gene for selection (selection gene), the preferred resistance gene and the most preferred antibiotic resistance gene is preferably positioned in the HCV-RNA construct after the HCV 5xe2x80x2to NTR, which means downstream from the 5xe2x80x2to NTR and upstream from the HCV reading frame. However, an insertion in the area of the 3xe2x80x2to NTR or another site of the HCV genome or subgenome, for example within the polyprotein, is also contemplated.
In another embodiment of the HCV-RNA construct according to the invention the marker gene for selection (selection gene), in particular an antibiotic resistance gene, is linked to the HCV-RNA or HCV genomic or subgenomic sequence via a ribozyme or a recognition site for a ribozyme.
This has the advantage, that after the selection of the cells, in which the HCV-RNA is replicating productively, the resistance gene in the obtained cell clones can be separated from the HCV subgenomic sequence through a ribozyme-dependent cleavage, namely by activating the inserted ribozyme or in the case of a construct with a recognition site for a ribozyme, by transfecting the ribozyme into the cells (for example through the transfection of a ribozyme construct or infection with a viral expression vector, into which the appropriate ribozyme has been inserted). By this means an authentic HCV genomic construct can be obtained without a resistance gene, which can then form authentic infectious virus particles.
Another preferred embodiment of the HCV-RNA construct according to the invention is characterized in that the construct has at least one integrated reporter gene.
In the following a reporter gene means any gene, whose presence can be easily detected with, in general, simple biochemical or also histochemical methods after being transferred into a target organism, which means a gene, that encodes for a protein, which can be easily and reliably detected and quantified in small amounts with the common measuring methods in the laboratory.
This variation of the HCV-RNA construct has the advantage that the extent of the replication of this construct can be easily and quickly measured with the methods common in the laboratory using the reporter gene product.
The reporter gene is preferably a gene from the group of the luciferase genes, the CAT gene (chloramphenicol acetyl transferase gene), the lacZ gene (beta galactosidase gene), the GFP gene (green fluorescence protein gene), the GUS gene (glucuronidase gene) or the SEAP gene (secreted alkaline phosphatase gene). This reporter gene and its products, namely the relevant reporter proteins, can be detected for example using fluorescence, chemiluminescence, colorimetric measurements or by means of immunological methods (for example ELISA).
A surrogate marker gene can also be considered as a reporter gene. In this context it includes those genes, which encode for cellular proteins, nucleic acids or generally for those functions, which are subject to variation depending on the replication of the virus, and which consequently are either suppressed or activated in the cells, in which the HCV or the HCV-RNA construct multiplies. This means, the suppression or activation of this function is a surrogate marker for the replication of the virus or the replication of the HCV-RNA construct.
The positions of the reporter genes and the marker gene for selection (selection gene) can be selected in such a way, that a fusion protein made from both genetic products will be expressed. This has the advantage that these two genes can be arranged in such a way in the HCV-RNA construct that their two expressed proteins are fused via a recognition sequence for a protease (for example ubiquitin) or via a self-cleaving peptide (for example the 2A protein of the Picornaviruses) at first and will be separated proteolytically later.
These two positions might as well lie apart from each other in such way, that both genetic products are separately expressed (for example in the order: marker or resistance genexe2x80x94internal ribosome binding sitexe2x80x94reporter gene).
In the case of the reporter gene one embodiment has proven to be particularly worthwhile, where the reporter gene is cloned into the open reading frame of the HCV genome or subgenome in such a way that it will only be transferred to an active form after proteolytic processing.
The cell culture system according to the invention can be used for various purposes in each of its embodiments. These include:
The detection of antiviral substances. This can include for example: organic compounds, which interfere directly or indirectly with viral growth (for example inhibitors of the viral proteases, the NS3 helicase, the NS5B RNA-dependent RNA polymerase), antisense oligonucleotides, which will hybridize to any target sequence in the HCV-RNA construct (for example the 5xe2x80x2to NTR) and will have an direct or indirect influence on the virus growth for example due to a reduction of translation of the HCV polyprotein or ribozymes, which cleave any HCV-RNA sequence and consequently impair virus replication.
The evaluation of any type of antiviral substances in the cell culture. These substances can be detected on the isolated purified enzyme for example with xe2x80x98rational drug designxe2x80x99 or xe2x80x98high-throughput screeningxe2x80x99. Evaluation means mainly the determination of the inhibitory features of the respective substance as well as its mode of action.
The identification of new targets of viral or cellular origin for a HCV specific antiviral therapy. If for example a cellular protein is essential for viral replication, the viral replication can also be influenced by inhibiting this cellular protein. The system according to the invention also enables the detection of these auxiliary factors.
The determination of drug resistance. It can be assumed that resistance to therapy occurs due to the high mutation rate of the HCV genome. This resistance, which is very important for the clinical approval of a substance, can be detected with the cell culture system according to the invention. Cell lines, in which the HCV-RNA construct or the HCV genome or subgenome replicates, are incubated with increasing concentrations of the relevant substance and the replication of the viral RNA is either determined by means of an introduced reporter gene or through the qualitative or quantitative detection of the viral nucleic acids or proteins. Resistance is given if no or a reduced inhibition of the replication can be observed with the normal concentration of the active substance. The nucleotide and amino acid replacements responsible for the therapy resistance can be determined by recloning the HCV-RNA (for example by the means of RT-PCR) and sequence analysis. By cloning the relevant replacement(s) into the original construct its causality for the resistance to therapy can be proven.
The production of authentic virus proteins (antigens) for the development and/or evaluation of diagnostics. The cell culture system according to the invention also allows the expression of HCV antigens in cell cultures. In principle these antigens can be used as the basis for diagnostic detection methods.
The production of HCV viruses and virus-like particles, in particular for the development or production of therapeutics and vaccines as well as for diagnostic purposes. Especially cell culture adapted complete HCV genomes, which could be produced by using the cell culture system according to the invention, are able to replicate in cell culture with high efficiency. These genomes have the complete functions of HCV and in consequence they are able to produce infectious viruses.
The HCV-RNA construct according to the invention by itself can also be used for various purposes in all its embodiments. This includes first of all:
The construction of attenuated hepatitis C viruses or HCV-like particles and their production in cell cultures:
Attenuated HCV or HCV-like particles can be created by accidental or purposefully introduced mutations, such as point mutations, deletions or insertions, which means viruses or virus-like particles with complete ability to replicate, but reduced or missing pathogenicity. These attenuated HCV or HCV-like particles can be used in particular as vaccine.
The construction of HCV-RNA constructs with integrated foreign genes, used for example as liver cell specific vector in gene therapy. Due to the distinctive liver cell tropism of the HCV and the possibility of replacing parts of the genome by heterologous sequences, HCV-RNA constructs can be produced, where for example the structural proteins can be replaced by a therapeutically effective gene. The HCV-RNA construct obtained in this way is introduced into cells preferably by means of transfection, which express the missing HCV functions, for example the structural proteins, in a constitutive or inducible way. Virus particles, carrying the HCV-RNA construct, can be created by means of this method known to the expert under the term xe2x80x98transcomplementationxe2x80x99. The particles obtained can preferably be used for the infection of liver cells. Within these the therapeutically effective foreign gene will be expressed and will consequently develop its therapeutic effect.
The detection of permissive cells, which means cells, in which a productive virus growth occurs. For this purpose either one of the HCV-RNA genomic constructs previously mentioned, which is able to form complete infectious viruses, or one of the HCV subgenomes previously mentioned, which according to the previously mentioned example will be transfected in a cell line first, which expresses the missing functions in a constitutive or inducible way, is used. In each case virus particles are created, which carry a resistance and/or reporter gene apart from the HCV sequence. In order to detect cells, where the HCV is able to replicate, these cells are infected with viruses generated in this way and subject to an antibiotic selection or they are examined depending on the HCV-RNA construct by means of determining the presence of the expression of the reporter gene. Because an antibiotic resistance or reporter gene expression can only be established, when HCV-RNA construct replicates, the cells detected in this way must be permissive. Almost any cell line or primary cell culture can be tested in regard to the permissivity and detected in this way.
The cell culture system according to the invention also permits targeted discovery of HCV-RNA constructs for which there is an increase in the efficiency of replication due to mutations. This occurs either by chance, in the context of HCV-RNA replication, or by targeted introduction into the construct. These mutations, leading to a change in the replication of the HCV-RNA construct, are known as adaptive mutations. The invention therefore also includes a method for obtaining cell culture adapted mutants of a HCV-RNA construct according to the invention following the above description, in which the mutants have increased replication efficiency compared to the original HCV-RNA construct. It further includes a method for the production of mutants of a HCV-RNA full-length genome or of a HCV-RNA subgenome or of any HCV-RNA construct with increased replication efficiency compared to the original HCV-RNA full-length genome or subgenome or HCV-RNA construct, as well as cell culture adapted mutants of HCV-RNA constructs, HCV-RNA full-length genomes and HCV subgenomes with increased replication efficiency compared to the original constructs, subgenomes or full-length genomes.
The method according to the invention for the production of cell culture adapted mutants of a HCV-RNA construct according to the invention, in which the mutants have increased replication efficiency compared to the HCV-RNA construct, is characterized in that a cell culture system according to the present invention, in which the transfected HCV specific genetic material is a HCV-RNA construct with a selection gene, is cultivated on/in the selection medium corresponding to the selection gene, that the cultivated cell clones are collected and that the HCV-RNA construct is isolated from these cell clones.
In certain embodiments, the isolated HCV-RNA constructs are passaged at least one more time, that is they are transfected in cells of a cell culture system according to the present invention to obtain the cell culture system according to the present invention, in which the transfected HCV specific genetic material is the isolated HCV-RNA construct with a selection gene, is cultivated on/in the selection medium corresponding to the selection gene, the cultivated cell clones are collected and the HCV-RNA constructs are thus isolated.
Using this process variation, the quantity of adaptive mutations and hence the degree of replication efficiency in the relevant HCV-RNA constructs can be increased even further.
The method according to the invention for the production of mutants of a HCV-RNA full-length genome or of a HCV-RNA subgenome or of any HCV-RNA construct with increased replication efficiency compared to the original HCV-RNA full-length genome or subgenome or HCV-RNA construct, has the following features. Using one of the two production methods presented above, a cell culture adapted mutant of a HCV-RNA construct is produced, isolated from the cells, cloned using the methods known in the art and sequenced. By comparing with the nucleotide and amino acid sequence of the original HCV-RNA construct, the type, number and position of the mutations is determined. These mutations are then introduced into an (isolated) HCV subgenome or full-length genome or any HCV-RNA construct, either by site-directed mutagenesis, or by exchange of DNA fragments containing the relevant mutations.
A test can be carried out to determine or verify which mutations actually are responsible for an alteration of replication efficiency, particularly an increase in replication. In this test the corresponding nucleotide and/or amino acid changes are introduced into the original HCV-RNA construct and the modified construct is then transfected in cell culture. If the introduced mutation actually leads to an increase in replication, then for a HCV-RNA construct with a selectable marker gene, the number of resistant cell clones in the artificially mutated construct should be noticeably higher compared to the untreated construct.
In the case of a construct with a reporter gene, the activity or quantity of the reporter should be noticeably higher for the artificially mutated construct compared to the untreated one.
The cell culture adapted HCV-RNA constructs with high replication efficiency according to the invention are characterized in that, through nucleotide or amino acid exchanges, they are derivable from a HCV-RNA construct of the present invention, and that they are obtainable using one of the two production processes presented above.
These cell culture adapted HCV-RNA constructs can be used to produce any HCV-RNA constructs or HCV full-length or subgenomes with increased replication efficiency. Both constructs with a selectable resistance gene and constructs without one or with a non-selectable reporter gene (e.g. luciferase) can be produced in this way, since replication of cell culture adapted HCV-RNA constructs can also be demonstrated in non-selected cells due to their high replication efficiency.
The cell culture adapted mutants of a HCV-RNA construct or HCV-RNA full-length genome or HCV subgenome with high replication efficiency compared to the original HCV-RNA construct or the original HCV full-length genome are characterized in that they are obtainable by a method in which the type and number of mutations in a cell culture adapted HCV-RNA construct are determined through sequence analysis and sequence comparison and these mutations are introduced into a HCV-RNA construct, particularly a HCV-RNA construct according to the present invention, or into an (isolated) HCV-RNA full-length genome, either by site-directed mutagenesis, or by exchange of DNA fragments containing the relevant mutations.
A group of preferred HCV-RNA constructs, HCV full-length genomes and HCV subgenomes with high and very high replication efficiency, which are consequently highly suitable for practical use is characterized in that it contains one, several or all of the amino acid or nucleic acid exchanges listed in table 3 and/or one or several of the following amino acid exchanges: 1283 argxe2x86x92gly , 1383 gluxe2x86x92ala , 1577 lysxe2x86x92arg , 1609 lysxe2x86x92glu , 1936 proxe2x86x92ser, 2163 gluxe2x86x92gly, 2330 lysxe2x86x92glu, 2442 ilexe2x86x92val. (The numbers refer to the amino acid positions of the polyprotein of the HCV isolate con1, see Table 1).
SEQ ID-NO: 1-3
Name: I389/Core-3xe2x80x2/wt
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-10842: HCV Polyprotein from Core up to nonstructural protein 5B
5. 1813-2385: HCV Core Protein; structural protein
6. 2386-2961: envelope protein 1 (E1); structural protein
7. 2962-4050: envelope protein 2 (E2); structural protein
8. 4051-4239: Protein p7
9. 4240-4890: nonstructural protein 2 (NS2); HCV NS2-3 Protease
10. 4891-6783: nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
11. 6784-6945: nonstructural protein 4A (NS4A); NS3 Protease cofactor
12. 6946-7728: nonstructural protein 4B (NS4B)
13. 7729-9069: nonstructural protein 5A (NS5A)
14. 9070-10842: nonstructural protein 5B (NS5B); RNA-dependent RNA-polymerase
15. 10846-11076: HCV 3xe2x80x2 non-translated region
SEQ ID-NO: 4-6
Name: I337/NS2-3xe2x80x2/wt
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1181: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1190-1800: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1801-8403: HCV Polyprotein from nonstructural protein 2 up to nonstructural protein 5B
5. 1801-2451: nonstructural protein 2 (NS2); HCV NS2-3 Protease
6. 2452-4344: nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
7. 4345-4506: nonstructural protein 4A (NS4A); NS3 Protease cofactor
8. 4507-5289: nonstructural protein 4B (NS4B)
9. 5290-6630: nonstructural protein 5A (NS5A)
10. 6631-8403: nonstructural protein 5B (NS5B); RNA-dependent RNA-polymerase
11. 8407-8637: HCV 3xe2x80x2 non-translated region
SEQ ID-NO: 7-9
Name: I389/NS3-3xe2x80x2/wt
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-7767: HCV Polyprotein from nonstructural protein 3 up to nonstructural protein 5B
5. 1813-3708: nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
6. 3709-3870: Nonstructural protein 4A (NS4A); NS3 Protease Cofactor
7. 3871-4653: Nonstructural protein 4B (NS4B)
8. 4654-5994: Nonstructural protein 5A (NS5A)
9. 5995-7767: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
10. 7771-8001: HCV 3xe2x80x2 non-translated Region
SEQ ID-NO: 10-12
Name: I337/NS3-3xe2x80x2/wt
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1181: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1190-1800: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1801-7758: HCV Polyprotein from Nonstructural protein 3 up to Nonstructural protein 5B
5. 1801-3696: Nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
6. 3697-3858: Nonstructural protein 4A (NS4A); NS3 Protease Cofactor
7. 3859-4641: Nonstructural protein 4B (NS4B)
8. 4642-5982: Nonstructural protein 5A (NS5A)
9. 5983-7755: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
10. 7759-7989: HCV 3xe2x80x2 non-translated Region
SEQ ID-NO: 13-15
Name: I389/NS2-3xe2x80x2/wt
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-8418: HCV Polyprotein from Nonstructural protein 2 up to Nonstructural protein 5B
5. 1813-2463: Nonstructural protein 2 (NS2); HCV NS2-3 Protease
6. 2464-4356: Nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
7. 4357-4518: Nonstructural protein 4A (NS4A); NS3 Protease Cofactor
8. 4519-5301: Nonstructural protein 4B (NS4B)
9. 5302-6642: Nonstructural protein SA (NS5A)
10. 6643-8415: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
11. 8419-8649: HCV 3xe2x80x2 non-translated Region
SEQ ID-NO: 16-18
Name: I389/NS3-3xe2x80x2/9-13F
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-7767: HCV Polyprotein from Nonstructural protein 3 up to Nonstructural protein 5B of the cell culture-adapted mutant 9-13F
5. 1813-3708: Nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
6. 3709-3870: Nonstructural protein 4A (NS4A); NS3,Protease Cofactor
7. 3871-4653: Nonstructural protein 4B (NS4B)
8. 4654-5994: Nonstructural protein 5A (NS5A)
9. 5995-7767: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
10. 7771-8001: HCV 3xe2x80x2 non-translated Region
SEQ ID-NO: 19-21
Name: I389/Core-3xe2x80x2/9-13F
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-10842: HCV Polyprotein from Core up to Nonstructural protein 5B of the cell culture-adapted mutant 9-13F
5. 1813-2385: HCV Core Protein; structural protein
6. 2386-2961: envelope protein 1 (E1); structural protein
7. 2962-4050: envelope protein 2 (E2); structural protein
8. 4051-4239: Protein p7
9. 4240-4890: Nonstructural protein 2 (NS2); HCV NS2-3 Protease
10. 4891-6783: Nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
11. 6784-6945: Nonstructural protein 4A (NS4A); NS3 Protease Cofactor
12. 6946-7728: Nonstructural protein 4B (NS4B)
13. 7729-9069: Nonstructural protein SA (NS5A)
14. 9070-10842: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
15. 10846-11076: HCV 3xe2x80x2 non-translated Region
SEQ ID-NO: 22-24
Name: I389/NS3-3xe2x80x2/5.1
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-7767: HCV Polyprotein from Nonstructural protein 3 up to Nonstructural protein 5B of the cell culture-adapted mutant 5.1
5. 1813-3708: Nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
6. 3709-3870: Nonstructural protein 4A (NS4A); NS3 Protease Cofactor
7. 3871-4653: Nonstructural protein 4B (NS4B)
8. 4654-5994: Nonstructural protein SA (NS5A)
9. 5995-7767: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
10. 7771-8001: HCV 3xe2x80x2 non-translated Region
SEQ ID-NO: 25-27
Name: I389/Core-3xe2x80x2/5.1
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-10842: HCV Polyprotein from Core up to Nonstructural protein 5B of the cell culture-adapted mutant 5.1
5. 1813-2385: HCV Core Protein; structural protein
6. 2386-2961: envelope protein 1 (E1); structural protein
7. 2962-4050: envelope protein 2 (E2); structural protein
8. 4051-4239: Protein p7
9. 4240-4890: Nonstructural protein 2 (NS2); HCV NS2-3 Protease
10. 4891-6783: Nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
11. 6784-6945: Nonstructural protein:4A (NS4A); NS3 Protease Cofactor
12. 6946-7728: Nonstructural protein 4B (NS4B)
13. 7729-9069: Nonstructural protein 5A (NS5A)
14. 9070-10842: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
15. 10846-11076: HCV 3xe2x80x2 non-translated Region
SEQ ID-NO: 28-30
Name: I389/NS3-3xe2x80x2/19
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-7767: HCV Polyprotein from Nonstructural protein 3 up to Nonstructural protein 5B of the cell culture-adapted mutant 19
5. 1813-3708: Nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
6. 3709-3870: Nonstructural protein 4A (NS4A); NS3 Protease Cofactor
7. 3871-4653: Nonstructural protein 4B (NS4B)
8. 4654-5994: Nonstructural protein 5A (NS5A)
9. 5995-7767: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
10. 7771-8001: HCV 3xe2x80x2 non-translated Region
SEQ ID-NO: 31-33
Name: I389/Core-3xe2x80x2/19
Composition (Nucleotide positions):
1. 1-341: HCV 5xe2x80x2 non-translated region
2. 342-1193: HCV Core Protein-Neomycin Phosphotransferase fusion protein; selectable Marker
3. 1202-1812: internal ribosome entry site from encephalomyokarditis virus; directs translation of the downstream located HCV open reading frame
4. 1813-10842: HCV Polyprotein from Core up to Nonstructural protein 5B of the cell culture-adapted mutant 19
5. 1813-2385: HCV Core Protein; structural protein
6. 2386-2961: envelope protein 1 (E1); structural protein
7. 2962-4050: envelope protein 2 (E2); structural protein
8. 4051-4239: Protein p7
9. 4240-4890: Nonstructural protein 2 (NS2); HCV NS2-3 Protease
10. 4891-6783: Nonstructural protein 3 (NS3); HCV NS3 Protease/Helicase
11. 6784-6945: Nonstructural protein 4A (NS4A); NS3 Protease Cofactor
12. 6946-7728: Nonstructural protein 4B (NS4B)
13. 7729-9069: Nonstructural protein 5A (NS5A)
14. 9070-10842: Nonstructural protein 5B (NS5B); RNA-dependent RNA-Polymerase
15. 10846-11076: HCV 3xe2x80x2 non-translated Region